Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

ABSTRACT

A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/819,866,filed on Mar. 14, 1997.

FIELD OF THE INVENTION

The present invention relates to a process of targeting the integrationof a desired exogenous DNA to a specific location within the genome or amammalian cell. More specifically, the invention describes a novelmethod for identifying a transcriptionally active target site (“hotspot”) in the mammalian genome, and inserting a desired DNA at this sitevia homologous recombination. The invention also optionally provides theability for gene amplification of the desired DNA at this location byco-integrating an amplifiable selectable marker, e.g., DHFR, incombination with the exogenous DNA. The invention additionally describesthe construction of novel vectors suitable for accomplishing the above,and further provides mammalian cell lines produced by such methods whichcontain a desired exogenous DNA integrated at a target hot spot.

BACKGROUND

Technology for expressing recombinant proteins in both prokaryotic andeukaryotic organisms is well established. Mammalian cells offersignificant advantages over bacteria or yeast for protein production,resulting from their ability to correctly assemble, glycosylate andpost-translationally modify recombinantly expressed proteins. Aftertransfection into the host cells, recombinant expression constructs canbe maintained as extrachromosomal elements, or may be integrated intothe host cell genome. Generation of stably transfected mammalian celllines usually involves the latter; a DNA construct encoding a gene ofinterest along with a drug resistance gene (dominant selectable marker)is introduced into the host cell, and subsequent growth in the presenceof the drug allows for the selection of cells that have successfullyintegrated the exogenous DNA. In many instances, the gene of interest islinked to a drug resistant selectable marker which can later besubjected to gene amplification. The gene encoding dihydrofolatereductase (DHFR) is most commonly used for this purpose. Growth of cellsin the presence of methotrexate, a competitive inhibitor of DHFR, leadsto increased DHFR production by means of amplification of the DHFR gene.As flanking regions of DNA will also become amplified, the resultantcoamplification of a DHFR linked gene in the transfected cell line canlead to increased protein production, thereby resulting in high levelexpression of the gene of interest.

While this approach has proven successful, there are a number ofproblems with the system because of the random nature of the integrationevent. These problems exist because expression levels are greatlyinfluenced by the effects of the local genetic environment at the genelocus, a phenomena well documented in the literature and generallyreferred to as “position effects” (for example, see Al-Shawi et al, Mol.Cell. Biol., 10:1192-1198 (1990); Yoshimura et al, Mol. Cell. Biol.,7:1296-1299 (1987)). As the vast majority of mammalian DNA is in atranscriptionally inactive state, random integration methods offer nocontrol over the transcriptional fate of the integrated DNA.Consequently, wide variations in the expression level of integratedgenes can occur, depending on the site of integration. For example,integration of exogenous DNA into inactive, or transcriptionally“silent” regions of the genome will result in little or no expression.By contrast integration into a transcriptionally active site may resultin high expression.

Therefore, when the goal of the work is to obtain a high level of geneexpression, as is typically the desired outcome of genetic engineeringmethods, it is generally necessary to screen large numbers oftransfectants to find such a high producing clone. Additionally, randomintegration of exogenous DNA into the genome can in some instancesdisrupt important cellular genes, resulting in an altered phenotype.These factors can make the generation of high expressing stablemammalian cell lines a complicated and laborious process.

Recently, our laboratory has described the use of DNA vectors containingtranslationally impaired dominant selectable markers in mammalian geneexpression. (This is disclosed in U.S. Ser. No. 08/147,696 filed Nov. 3,1993, recently allowed).

These vectors contain a translationally impaired neomycinphosphotransferase (neo) gene as the dominant selectable marker,artificially engineered to contain an intron into which a DHFR genealong with a gene or genes of interest is inserted. Use of these vectorsas expression constructs has been found to significantly reduce thetotal number of drug resistant colonies produced, thereby facilitatingthe screening procedure in relation to conventional mammalian expressionvectors. Furthermore, a significant percentage of the clones obtainedusing this system are high expressing clones. These results areapparently attributable to the modifications made to the neo selectablemarker. Due to the translational impairment of the neo gene, transfectedcells will not produce enough neo protein to survive drug selection,thereby decreasing the overall number of drug resistant colonies.Additionally, a higher percentage of the surviving clones will containthe expression vector integrated into sites in the genome where basaltranscription levels are high, resulting in overproduction of neo,thereby allowing the cells to overcome the impairment of the neo gene.Concomitantly, the genes of interest linked to neo will be subject tosimilar elevated levels of transcription. This same advantage is alsotrue as a result of the artificial intron created within neo; survivalis dependent on the synthesis of a functional neo gene, which is in turndependent on correct and efficient splicing of the neo introns.Moreover, these criteria are more likely to be met if the vector DNA hasintegrated into a region which is already highly transcriptionallyactive.

Following integration of the vector into a transcriptionally activeregion, gene amplification is performed by selection for the DHFR gene.Using this system, it has been possible to obtain clones selected usinglow levels of methotrexate (50 nM), containing few (<10) copies of thevector which secrete high levels of protein (>55 pg/cell/day).Furthermore, this can be. achieved in a relatively short period of time.However, the success in amplification is variable. Sometranscriptionally active sites cannot be amplified and therefore thefrequency and extent of amplification from a particular site is notpredictable.

Overall, the use of these translationally impaired vectors represents asignificant improvement over other methods of random integration.However, as discussed, the problem of lack of control over theintegration site remains a significant concern.

One approach to overcome the problems of random integration is by meansof gene targeting, whereby the exogenbus DNA is directed to a specificlocus within the host genome. The exogenous DNA is inserted by means ofhomologous recombination occurring between sequences of DNA in theexpression vector and the corresponding homologous sequence in thegenome. However, while this type of recombination occurs at a highfrequency naturally in yeast and other fungal organisms, in highereukaryotic organisms it is an extremely rare event. In mammalian cells,the frequency of homologous versus non-homologous (random integration)recombination is reported to range from 1/100 to 1/5000 (for example,see Capecchi, Science, 244:1288-1292 .(1989); Morrow and Kucherlapati,Curr. Op. Biotech., 4:577-582 (1993)).

One of the earliest reports describing homologous recombination inmammalian cells comprised an artificial system created in mousefibroblasts (Thomas et al, Cell, 44:419-428 (1986)). A cell linecontaining a mutated, non-functional version of the neo gene integratedinto the host genome was created, and subsequently targeted with asecond non-functional copy of neo containing a different mutation.Reconstruction of a functional neo gene could occur only by genetargeting. Homologous recombinants were identified by selecting for G418resistant cells, and confirmed by analysis of genomic DNA isolated fromthe resistant clones.

Recently, the use of homologous recombination to replace the heavy andlight immunoglobulin genes at endogenous loci in antibody secretingcells has been reported. (U.S. Pat. No. 5,202,238, Fell et al, (1993).)However, this particular approach is not widely applicable, because itis limited to the production of immunoglobulins in cells whichendogenously express immunoglobulins, e.g., B cells and myeloma cells.Also, expression is limited to single copy gene levels becauseco-amplification after homologous recombination is not included. Themethod is further complicated by the fact that two separate integrationevents are required to produce a functional immunoglobulin: one for thelight chain gene followed by one for the heavy chain gene.

An additional example of this type of system has been reported in NS/0cells, where recombinant immunoglobulins are expressed by homologousrecombination into the immunoglobulin gamma 2A locus (Hollis et al,international patent application # PCT/IB95 (00014).) Expression levelsobtained from this site were extremely high—on the order of 20pg/cell/day from a single copy integrant. However, as in the aboveexample, expression is limited to this level because an amplifiable geneis not contegrated in this system. Also, other researchers have reportedaberrant glycosylation of recombinant proteins expressed in NS/0 cells(for example, see Flesher et al, Biotech. and Bioeng., 48:399-407(1995)), thereby limiting the applicability of this approach.

The cre-loxP recombination system from bacteriophage P1 has recentlybeen adapted and used as a means of gene targeting in eukaryotic cells.Specifically, the site specific integration of exogenous DNA into theChinese hamster ovary (CHO) cell genome using cre recombinase and aseries of lox containing vectors have been described. (Fukushige andSauer, Proc. Natl. Acad. Sci. USA, 89:7905-7909 (1992).) This system isattractive in that it provides for reproducible expression at the samechromosomal location. However, no effort was made to identify achromosomal site from which gene expression is optimal, and as in theabove example, expression is limited to single copy levels in thissystem. Also, it is complicated by the fact that one needs to providefor expression of a functional recombinase enzyme in the mammalian cell.

The use of homologous recombination between an introduced DNA sequenceand its endogenous chromosomal locus has also been reported to provide auseful means of genetic manipulation in mammalian cells, as well as inyeast cells. (See e.g., Bradley et al, Meth. Enzymol., 223:855-879(1993); Capecchi, Science, 244:1288-1292 (1989); Rothstein et al, Meth.Enzymol., 194:281-301 (1991)). To date, most mammalian gene targetingstudies have been directed toward gene disruption (“knockout”) orsite-specific mutagenesis of selected target gene loci in mouseembryonic stem (ES) cells. The creation of these “knockout” mouse modelshas enabled scientists to examine specific structure-function issues andexamine the biological importance of a myriad of mouse genes. This fieldof research also has important implications in terms of potential genetherapy applications.

Also, vectors have recently been reported by Cell-tech (Kent, U.K.)which purportedly are targeted to transcriptionally active sites in NSOcells, which do not require gene amplification (Peakman et al, Hum.Antibod. Hybridomas, 5:65-74 (1994)). However, levels of immunoglobulinsecretion in these unamplified cells have not been reported to exceed 20pg/cell/day, while in amplified CHO cells, levels as high as 100pg/cell/day can be obtained (Id.).

It would be highly desirable to develop a gene targeting system whichreproducibly provided for the integration of exogenous DNA into apredetermined site in the genome known to be transcriptionally active.Also, it would be desirable if such a gene targeting system wouldfurther facilitate co-amplification of the inserted DNA afterintegration. The design of such a system would allow for thereproducible and high level expression of any cloned gene of interest ina mammalian cell, and undoubtedly would be of significant interest tomany researchers.

In this application, we provide a novel mammalian expression system,based on homologous recombination occurring between two artificialsubstrates contained in two different vectors. Specifically, this systemuses a combination of two novel mammalian expression vectors, referredto as a “marking” vector and a “targeting” vector.

Essentially, the marking vector enables the identification and markingof a site in the mammalian genome which is transcriptionally active,i.e., a site at which gene expression levels are high. This site can beregarded as a “hot spot” in the genome. After integration of the markingvector, the subject expression system enables another DNA to beintegrated at this site, i.e., the targeting vector, by means ofhomologous recombination occurring between DNA sequences common to bothvectors. This system affords significant advantages over otherhomologous recombination systems.

Unlike most other homologous systems employed in mammalian cells, thissystem exhibits no background. Therefore, cells which have onlyundergone random integration of the vector do not survive the selection.Thus, any gene of interest cloned into the targeting plasmid isexpressed at high levels from the marked hot spot. Accordingly, thesubject method of gene expression substantially or completely eliminatesthe problems inherent to systems of random integration, discussed indetail above. Moreover, this system provides reproducible and high levelexpression of any recombinant protein at the same transcriptionallyactive site in the mammalian genome. In addition, gene amplification maybe effected at this particular transcriptionally active site byincluding an amplifiable dominant selectable marker (e.g. DHFR) as partof the marking vector.

OBJECTS OF THE INVENTION

Thus, it is an object of the invention to provide an improved method fortargeting a desired DNA to a specific site in a mammalian cell.

It is a more specific object of the invention to provide a novel methodfor targeting a desired DNA to a specific site in a mammalian cell viahomologous recombination.

It is another specific object of the invention to provide novel vectorsfor achieving site specific integration of a desired DNA in a mammaliancell.

It is still another object of the invention to provide novel mammaliancell lines which contain a desired DNA integrated at a predeterminedsite which provides for high expression.

It is a more specific object of the invention to provide a novel methodfor achieving site specific integration of a desired DNA in a Chinesehamster ovary (CHO) cell.

It is another more specific object of the invention to provide a novelmethod for integrating immunoglobulin genes, or any other genes, inmammalian cells at predetermined chromosomal sites that provide for highexpression.

It is another specific object of the invention to provide novel vectorsand vector combinations suitable for integrating immunoglobulin genesinto mammalian cells at predetermined sites that provide for highexpression.

It is another object of the invention to provide mammalian cell lineswhich contain immunoglobulin genes integrated at predetermined sitesthat provide for high expression.

It is an even more specific object of the invention to provide a novelmethod for integrating immunoglobulin genes into CHO cells that providefor high expression, as well as novel vectors and vector combinationsthat provide for such integration of immunoglobulin genes into CHOcells.

In addition, it is a specific object of the invention to provide novelCHO cell lines which contain immunoglobulin genes integrated atpredetermined sites that provide for high expression, and have beenamplified by methotrexate selection to secrete even greater amounts offunctional immunoglobulins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a map of a marking plasmid according to the inventionreferred to as Desmond. The plasmid is shown in circular form (1 a) aswell as a linearized version used for transfection (1 b).

FIG. 2( a) shows a map of a targeting plasmid referred to “Molly”. Mollyis shown here encoding the anti-CD20 immunoglobulin genes, expression ofwhich is described in Example 1.

FIG. 2( b) shows a linearized version of Molly, after digestion with therestriction enzymes Kpn1 and Pac1. This linearized form was used fortransfection.

FIG. 3 depicts the potential alignment between Desmond sequencesintegrated into the CHO genome, and incoming targeting Molly sequences.One potential arrangement of Molly integrated into Desmond afterhomologous recombination is also presented.

FIG. 4 shows a Southern analysis of single copy Desmond clones. Samplesare as follows:

Lane 1: λHindIII DNA size markerLane 2: Desmond clone 10F3Lane 3: Desmond clone 10C12Lane 4: Desmond clone 15C9Lane 4: Desmond clone 14B5Lane 6: Desmond clone 9B2

FIG. 5 shows a Northern analysis of single copy Desmond clones. Samplesare as follows: Panel A: northern probed with CAD and DHFR probes, asindicated on the figure. Panel B: duplicate northern, probed with CADand HisD probes, as indicated. The RNA samples loaded in panels A and Bare as follows:

Lane 1: clone 9B2, lane 2; clone 10C12, lane 3; clone 14B5, lane 4;clone 15C9, lane 5; control RNA from CHO transfected with a HisD andDHFR containing plasmid, lane 6; untransfected CHO.

FIG. 6 shows a Southern analysis of clones resulting from the homologousintegration of Molly into Desmond. Samples are as follows:

Lane 1: λHindIII DNA size markers, Lane 2: 20F4, lane 3; 5F9, lane 4;21C7, lane 5; 24G2, lane 6; 25E1, lane 7; 28C9, lane 8; 29F9, lane 9;39G11, lane 10; 42F9, lane 11; 50G10, lane 12; Molly plasmid DNA,linearized with BglII(top band) and cut with BglII and KpnI (lowerband), lane 13; untransfected Desmond.

FIGS. 7A through 7G contain the Sequence Listing for Desmond.

FIGS. 8A through 8I contain the Sequence Listing for Molly-containinganti-CD20.

FIG. 9 contains a map of the targeting plasmid, “Mandy,” shown hereencoding anti-CD23 genes, the expression of which is disclosed inExample 5.

FIGS. 10A through 10N contain the sequence listing of “Mandy” containingthe anti-CD23 genes as disclosed in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel method for integrating a desiredexogenous DNA at a target site within the genome of a mammalian cell viahomologous recombination. Also, the invention provides novel vectors forachieving the site specific integration of a DNA at a target site in thegenome of a mammalian cell.

More specifically, the subject cloning method provides for site specificintegration of a desired DNA in a mammalian cell by transfection of suchcell with a “marker plasmid” which contains a unique sequence that isforeign to the mammalian cell genome and which provides a substrate forhomologous recombination, followed by transfection with a “targetplasmid” containing a sequence which provides for homologousrecombination with the unique sequence contained in the marker plasmid,and further comprising a desired DNA that is to be integrated into themammalian cell. Typically, the integrated DNA will encode a protein ofinterest, such as an immunoglobulin or other secreted mammalianglycoprotein.

The exemplified homologous recombination system uses the neomycinphosphotransferase gene as a dominant selectable marker. This particularmarker was utilized based on the following previously publishedobservations;

(i) the demonstrated ability to target and restore function to a mutatedversion of the neo gene (cited earlier) and

(ii) our development of translationally impaired expression vectors, inwhich the neo gene has been artificially created as two exons with agene of interest inserted in the intervening intron; neo exons arecorrectly spliced and translated in vivo, producing a functional proteinand thereby conferring G418 resistance on the resultant cell population.In this application, the neo gene is split into three exons. The thirdexon of neo is present on the “marker” plasmid and becomes integratedinto the host cell genome upon integration of the marker plasmid intothe mammalian cells. Exons 1 and 2 are present on the targeting plasmid,and are separated by an intervening intron into which at least one geneof interest is cloned. Homologous recombination of the targeting vectorwith the integrated marking vector results in correct splicing of allthree exons of the neo gene and thereby expression of a functional neoprotein (as determined by selection for G418 resistant colonies). Priorto designing the current expression system, we had experimentally testedthe functionality of such a triply spliced neo construct in mammaliancells. The results of this control experiment indicated that all threeneo exons were properly spliced and therefore suggested the feasibilityof the subject invention.

However, while the present invention is exemplified using the neo gene,and more specifically a triple split neo gene, the general methodologyshould be efficacious with other dominant selectable markers.

As discussed in greater detail infra, the present invention affordsnumerous advantages to conventional gene expression methods, includingboth random integration and gene targeting methods. Specifically, thesubject invention provides a method which reproducibly allows forsite-specific integration of a desired DNA into a transcriptionallyactive domain of a mammalian cell. Moreover, because the subject methodintroduces an artificial region of “homology” which acts as a uniquesubstrate for homologous recombination and the insertion of a desiredDNA, the efficacy of subject invention does not require that the cellendogenously contain or express a specific DNA. Thus, the method isgenerically applicable to all mammalian cells, and can be used toexpress any type of recombinant protein.

The use of a triply spliced selectable marker, e.g., the exemplifiedtriply spliced neo construct, guarantees that all G418 resistantcolonies produced will arise from a homologous recombination event(random integrants will not produce a functional neo gene andconsequently will not survive G418 selection). Thus, the subjectinvention makes it easy to screen for the desired homologous event.Furthermore, the frequency of additional random integrations in a cellthat has undergone a homologous recombination event appears to be low.

Based on the foregoing, it is apparent that a significant advantage ofthe invention is that it substantially reduces the number of coloniesthat need be screened to identify high producer clones, i.e., cell linescontaining a desired DNA which secrete the corresponding protein at highlevels. On average, clones containing integrated desired DNA may beidentified by screening about 5 to 20 colonies (compared to severalthousand which must be screened when using standard random integrationtechniques, or several hundred using the previously described intronicinsertion vectors) Additionally, as the site of integration waspreselected and comprises a transcriptionally active domain, allexogenous DNA expressed at this site should produce comparable, i.e.high levels of the protein of interest.

Moreover, the subject invention is further advantageous in that itenables an amplifiable gene to be inserted on integration of the markingvector. Thus, when a desired gene is targeted to this site via.homologous recombination, the subject invention allows for expression ofthe gene to be further enhanced by gene amplification. In this regard,it has been reported in from the literature that different genomic sitese different capacities for gene amplification (Meinkoth et al, Mol. CellBiol., 7:1415-1424 (1987)). Therefore, this technique is furtheradvantageous as it allows for the placement of a desired gene ofinterest at a specific site that is both transcriptionally active andeasily amplified. Therefore, this should significantly reduce the amountof time required to isolate such high producers.

Specifically, while conventional methods for the construction of highexpressing mammalian cell lines can take 6 to 9 months, the presentinvention allows for such clones to be isolated on average after onlyabout 3-6 months. This is due to the fact that conventionally isolatedclones typically must be subjected to at least three rounds of drugresistant gene amplification in order to reach satisfactory levels ofgene expression. As the homologously produced clones are generated froma preselected site which is a high expression site, fewer rounds ofamplification should be required before reaching a satisfactory level ofproduction.

Still further, the subject invention enables the reproducible selectionof high producer clones wherein the vector is integrated at low copynumber, typically single copy. This is advantageous as it enhances thestability of the clones and avoids other potential adverse-side-effectsassociated with high copy number. As described supra, the subjecthomologous recombination system uses the combination of a “markerplasmid” and a “targeting plasmid” which are described in more detailbelow.

The “marker plasmid” which is used to mark and identify atranscriptionally hot spot will comprise at least the followingsequences:

(i) a region of DNA that is heterologous or unique to the genome of themammalian cell, which functions as a source of homology, allows forhomologous recombination (with a DNA contained in a second targetplasmid). More specifically, the unique region of DNA (i) will generallycomprise a bacterial, viral, yeast synthetic, or other DNA which is notnormally present in the mammalian cell genome and which further does notcomprise significant homology or sequence identity to DNA contained inthe genome of the mammalian cell. Essentially, this sequence should besufficiently different to mammalian DNA that it will not significantlyrecombine with the host cell genome via homologous recombination. Thesize of such unique DNA will generally be at least about 2 to 10kilobases in size, or higher, more preferably at least about 10 kb, asseveral other investigators have noted an increased frequency oftargeted recombination as the size of the homology region is increased(Capecchi, Science, 244:1288-1292 (1989)).

The upper size limit of the unique DNA which acts as a site forhomologous recombination with a sequence in the second target vector islargely dictated by potential stability constraints (if DNA is too largeit may not be easily integrated into a chromosome and the difficultiesin working with very large DNAs.

(ii) a DNA including a fragment of a selectable marker DNA, typically anexon of a dominant selectable marker gene. The only essential feature ofthis DNA is that it not encode a functional selectable marker proteinunless it is expressed in association with a sequence contained in thetarget plasmid. Typically, the target plasmid will comprise theremaining exons of the dominant selectable marker gene (those notcomprised in “targeting” plasmid). Essentially, a functional selectablemarker should only be produced if homologous recombination occurs(resulting in the association and expression of this marker DNA (i)sequence together with the portion(s) of the selectable marker DNAfragment which is (are) contained in the target plasmid).

As noted, the current invention exemplifies the use of the neomycinphosphotransferase gene as the dominant selectable marker which is“split” in the two vectors. However, other selectable markers shouldalso be suitable, e.g., the Salmonella histidinol dehydrogenase gene,hygromycin phosphotransferase gene, herpes simplex virus thymidinekinase gene, adenosine deaminase gene, glutamine synthetase gene andhypoxanthine-guanine phosphoribosyl transferase gene.

(iii) a DNA which encodes a functional selectable marker protein, whichselectable marker is different from the selectable marker DNA (ii). Thisselectable marker provides for the successful selection of mammaliancells wherein the marker plasmid is successfully integrated into thecellular DNA. More preferably, it is desirable that the marker plasmidcomprise two such dominant selectable marker DNAS, situated at oppositeends of the vector. This is advantageous as it enables integrants to beselected using different selection agents and further enables cellswhich contain the entire vector to be selected. Additionally, one markercan be an amplifiable marker to facilitate gene dominant selectablemarker listed in (ii) can be used as well as others generally known inthe art.

Moreover, the marker plasmid may optionally further comprise a rareendonuclease restriction site. This is potentially desirable as this mayfacilitate cleavage. If present, such rare restriction site should besituated close to the middle of the unique region that acts as asubstrate for homologous recombination. Preferably such sequence will beat least about 12 nucleotides. The introduction of a double strandedbreak by similar methodology has been reported to enhance the frequencyof homologous recombination. (Choulika et al, Mol. Cell. Biol.,15:1968-1973 (1995)). However, the presence of such sequence is notessential.

The “targeting plasmid” will comprise at least the following sequences:

(1) the same unique region of DNA contained in the marker plasmid or onehaving sufficient homology or sequence identity therewith that said DNAis capable of combining via homologous recombination with the uniqueregion (i) in the marker plasmid. Suitable types of DNAs are describedsupra in the description of the unique region of DNA (1) in the markerplasmid.

(2) The remaining exons of the dominant selectable marker, one exon ofwhich is included as (ii) in the marker plasmid listed above. Theessential features of this DNA fragment is that it result in afunctional (selectable) marker protein only if the target plasmidintegrates via homologous recombination (wherein such recombinationresults in the association of this DNA with the other fragment of theselectable marker DNA contained in the marker plasmid) and further thatit allow for insertion of a desired exogenous DNA. Typically, this DNAwill comprise the remaining exons of the selectable marker DNA which areseparated by an intron. For example, this DNA may comprise the first twoexons of the neo gene and the marker plasmid may comprise the third exon(back third of neo).

(3) The target plasmid will also comprise a desired DNA, e.g., oneencoding a desired polypeptide, preferably inserted within theselectable marker DNA fragment contained in the plasmid. Typically, theDNA will be inserted in an intron which is comprised between the exonsof the selectable marker DNA. This ensures that the desired DNA is alsointegrated if homologous recombination of the target plasmid and themarker plasmid occurs. This intron may be naturally occurring or it maybe engineered into the dominant selectable marker DNA fragment.

This DNA will encode any desired protein, preferably one havingpharmaceutical or other desirable properties. Most typically the DNAwill encode a mammalian protein, and in the current examples provided,an immunoglobulin or an immunoadhesin. However the invention is not inany way limited to the production of immunoglobulins.

As discussed previously, the subject cloning method is suitable for anymammalian cell as it does not require for efficacy that any specificmammalian sequence or sequences be present. In general, such mammaliancells will comprise those typically used for protein expression, e.g.,CHO cells, myeloma cells, COS cells, BHK cells, Sp2/0 cells, NIH 3T3 andHeLa cells. In the. examples which follow, CHO cells were utilized. Theadvantages thereof include the availability of suitable growth medium,their ability to crow efficiently and to high density in culture, andtheir ability to express mammalian proteins such as immunoglobulins inbiologically active form.

Further, CHO cells were selected in large part because of previous usageof such cells by the inventors for the expression of immunoglobulins(using the translationally impaired dominant selectable markercontaining vectors described previously). Thus, the present laboratoryhas considerable experience in using such cells for expression. However,based on the examples. which follow, it is reasonable to expect similarresults will be obtained with other mammalian cells.

In general, transformation or transfection of mammalian cells accordingto the subject invention will be effected according to conventionalmethods. So that the invention may be better understood, theconstruction of exemplary vectors and their usage in producingintegrants is described in the examples below.

EXAMPLE 1 Design and Preparation of Marker and Targeting Plasmid DNAVectors

The marker plasmid herein referred to as “Desmond” was assembled fromthe following DNA elements:

(a) Murine dihydrofolate reductase gene (DHFR), incorporated into atranscription cassette, comprising the mouse beta globin promoter 5″ tothe DHFR start site, and bovine growth hormone poly adenylation signal3″ to the stop codon. The DHFR transcriptional cassette was isolatedfrom TCAE6, an expression vector created previously in this laboratory(Newman et al, 1992, Biotechnology, 10:1455-1460).

(b) E. coli β-galactosidase gene—commercially available, obtained fromPromega as pSV-b-galactosidase control vector, catalog # E1081.

(c) Baculovirus DNA, commercially available, purchased from Clontech aspBAKPAK8, cat # 6145-1.

(d) Cassette comprising promoter and enhancer elements fromCytomegalovirus and SV40 virus. The cassette was generated by PCR usinga derivative of expression vector TCAE8 (Reff et al, Blood, 83:435-445(1994)). The enhancer cassette was inserted within the baculovirussequence, which was first modified by the insertion of a multiplecloning site.

(e) E. coli GUS (glucuronidase) gene, commercially available, purchasedfrom Clontech as pB101, cat. # 6017-1.

(f) Firefly luciferase gene, commercially available, obtained fromPromega as pGEM-Luc (catalog # E1541).

(g) S. typhimurium histidinol dehydrogenase gene (HisD). This gene wasoriginally a gift from (Donahue et el, Gene, 18:47-59 (1982)), and hassubsequently been incorporated into a transcription cassette comprisingthe mouse beta globin major promoter 5′ to the gene, and the SV40polyadenylation signal 3′ to the gene.

The DNA elements described in (a)-(g) were combined into a pBR derivedplasmid backbone to produce a 7.7 kb contiguous stretch of DNA referredto in the attached figures as “homology”. Homology in this sense refersto sequences of DNA which are not part of the mammalian genome and areused to promote homologous recombination between transfected plasmidssharing the same homology DNA sequences.

(h) Neomycin phosphotransferase gene from TN5 (Davis and Smith, Ann.Rev. Micro., 32:469-518 (1978)). The complete neo gene was subclonedinto pBluescript SK-(Stratagene catalog # 212205) to facilitate geneticmanipulation. A synthetic linker was then inserted into a unique Pst1site occurring across the codons for amino acid 51 and 52 of neo. Thislinker encoded the necessary DNA elements to create an artificial splicedonor site, intervening intron and splice acceptor site within the neogene, thus creating two separate exons, presently referred to as neoexon 1 and 2. Neo exon 1 encodes the first 51 amino acids of neo, whileexon 2 encodes the remaining 203 amino acids plus the stop codon of theprotein A Not1 cloning site was also created within the intron.

Neo exon 2 was further subdivided to produce neo exons 2 and 3. This wasachieved as follows: A set of PCR primers were designed to amplify aregion of DNA encoding neo exon 1, intron and the first 111 2/3 aminoacids of exon2. The 3′ PCR primer resulted in the introduction of a new5′ splice site immediately after the second nucleotide of the codon foramino acid 111 in exon 2, therefore generating a new smaller exon 2. TheDNA fragment now encoding the original exon 1, intron and new exon 2 wasthen subcloned and propagated in a pBR based vector. The remainder ofthe original exon 2 was used as a template for another round of PCRamplification, which generated “exon3”. The 5′ primer for this round ofamplification introduced a new splice acceptor site at the 5′ side ofthe newly created exon 3, i.e. before the final nucleotide of the codonfor amino acid 111. The resultant 3 exons of neo encode the followinginformation: exon 1—the first 51 amino acids of neo; exon 2—the next 1112/3 amino acids, and exon 3 the final 91 1/3 amino acids plus thetranslational stop codon of the neo gene.

Neo exon 3 was incorporated along with the above mentioned DNA elementsinto the marking plasmid “Desmond”. Neo exons 1 and 2 were incorporatedinto the targeting plasmid “Molly”. The Not1 cloning site created withinthe intron between exons 1 and 2 was used in subsequent cloning steps toinsert genes of interest into the targeting plasmid.

A second targeting plasmid “Mandy” was also generated. This plasmid isalmost identical to “Molly” (some restriction sites on the vector havebeen changed) except that the original HisD and DHFR genes contained in“Molly” were inactivated. These changes were incorporated because theDesmond cell line was no longer being cultured in the presence ofHistidinol, therefore it seemed unnecessary to include a second copy ofthe HisD gene. Additionally, the DHFR gene was inactivated to ensurethat only a single DHFR gene, namely the one present in the Desmondmarked site, would be amplifiable in any resulting cell lines. “Mandy”was derived from “Molly” by the following modifications:

(i) A synthetic linker was inserted in the middle of the DHFR codingregion. This linker created a stop codon and shifted the remainder ofthe DHFR coding region out of frame, therefore rendering the genenonfunctional.

(ii) A portion of the HisD gene was deleted and replaced with a PCRgenerated HisD fragment lacking the promoter and start codon of thegene.

FIG. 1 depicts the arrangement of these DNA elements in the markerplasmid “Desmond”. FIG. 2 depicts the arrangement of these elements inthe first targeting plasmid, “Molly”. FIG. 3 illustrates the possiblearrangement in the CHO genome, of the various DNA elements aftertargeting and integration of Molly DNA into Desmond marked CHO cells.FIG. 9 depicts the targeting plasmid “Mandy.”

Construction of the marking and targeting plasmids from the above listedDNA elements was carried out following conventional cloning techniques(see, e.g., Molecular Cloning, A Laboratory Manual, J. Sambrook et al,1987, Cold Spring Harbor Laboratory Press, and Current Protocols inMolecular Biology, F. M. Ausubel et al, eds., 1987, John Wiley andSons). All plasmids were propagated and maintained in E. coli XLI blue(Stratagene, cat. # 200236). Large scale plasmid preparations wereprepared using Promega Wizard Maxiprep DNA Purification System®,according to the manufacturer's directions.

EXAMPLE 2 Construction of a Marked CHO Cell Line 1. Cell Culture andTransfection Procedures to Produced Marked CHO Cell Line

Marker plasmid DNA was linearized by digestion overnight at 37° C. withBst1107I. Linearized vector was ethanol precipitated and resuspended insterile TE to a concentration of 1 mg/ml. Linearized vector wasintroduced into DHFR-Chinese hamster ovary cells (CHO cells) DG44 cells(Urlaub et al, Som. Cell and Mol. Gen., 12:555-566 (1986)) byelectroporation as follows.

Exponentially growing cells were harvested by centrifugation, washedonce in ice cold SBS (sucrose buffered solution, 272 mM sucrose, 7 mMsodium phosphate, pH 7.4, 1 mM magnesium chloride) then resuspended inSBS to a concentration of 10⁷ cells/ml. After a 15 minute incubation onice, 0.4 ml of the cell suspension was mixed with 40 μg linearized DNAin a disposable electroporation cuvette. Cells were shocked using a BTXelectrocell manipulator (San Diego, Calif.) set at 230 volts, 400microfaraday capacitance, 13 ohm resistance. Shocked cells were thenmixed with 20 ml of prewarmed CHO growth media (CHO-S-SFMII, Gibco/BRL,catalog # 31033-012) and plated in 96 well tissue culture plates. Fortyeight hours after electroporation, plates were fed with selection media(in the case of transfection with Desmond, selection media isCHO-S-SFMII without hypoxanthine or thymidine, supplemented with 2 mMHistidinol (Sigma catalog # H6647)). Plates were maintained in selectionmedia for up to 30 days, or until some of the wells exhibited cellgrowth. These cells were then removed from the 96 well plates andexpanded ultimately to 120 ml spinner flasks where they were maintainedin selection media at all times.

EXAMPLE 3 Characterization of Marked CHO Cell Lines (a) SouthernAnalysis

Genomic DNA was isolated from all stably growing Desmond marked CHOcells. DNA was isolated using the Invitrogen Easy® DNA kit, according tothe manufacturer's directions. Genomic DNA was then digested withHindIII overnight at 37° C., and subjected to Southern analysis using aPCR generated digoxygenin labelled probe specific to the DHFR gene.Hybridizations and washes were carried out using Boehringer Mannheim'sDIG easy hyb (catalog # 1603 558) and DIG Wash and Block Buffer Set(catalog # 1585 762) according to the manufacturer's directions. DNAsamples containing a single band hybridizing to the DHFR probe wereassumed to be Desmond clones arising from a single cell which hadintegrated a single copy of the plasmid. These clones were retained forfurther analysis. Out of a total of 45 HisD resistant cell linesisolated, only 5 were single copy integrants. FIG. 4 shows a Southernblot containing all 5 of these single copy Desmond clones. Clone namesare provided in the figure legend.

(b) Northern Analysis

Total RNA was isolated from all single copy Desmond clones using TRIzolreagent (Gibco/BRL cat # 15596-026) according to the manufacturer'sdirections. 10-20 μg RNA from each clone was analyzed on duplicateformaldehyde gels. The resulting blots were probed with PCR generateddigoxygenin labelled DNA probes to (i) DHFR message, (ii) HisD messageand (iii) CAD message. CAD is a trifunctional protein involved inuridine biosynthesis (Wahl et al, J. Biol. Chem., 254, 17:8679-8689(1979)), and is expressed equally in all cell types. It is used here asan internal control to help quantitate RNA loading. Hybridizations andwashes were carried out using the above mentioned Boehringer Mannheimreagents. The results of the Northern analysis are shown in FIG. 5. Thesingle copy Desmond clone exhibiting the highest levels of both the HisD and DHFR message is clone 15C9, shown in lane 4 in both panels of thefigure. This clone was designated as the “marked cell line” and used infuture targeting experiments in CHO, examples of which are presented inthe following sections.

EXAMPLE 4 Expression of Anti-CD20 Antibody in Desmond Marked CHO Cells

C2B8, a chimeric antibody which recognizes B-cell surface antigen CD20,has been cloned and expressed previously in our laboratory. (Reff et al,Blood, 83:434-45 (1994)). A 4.1 kb DNA fragment comprising the C2B8light and heavy chain genes, along with the necessary regulatoryelements (eukaryotic promoter and polyadenylation signals) was insertedinto the artificial intron created between exons 1 and 2 of the neo genecontained in a pBR derived cloning vector. This newly generated 5 kb DNAfragment (comprising neo exon 1, C2B8 and neo exon 2) was excised andused to assemble the targeting plasmid Molly. The other DNA elementsused in the construction of Molly are identical to those used toconstruct the marking plasmid Desmond, identified previously. A completemap of Molly is shown in FIG. 2.

The targeting vector Molly was linearized prior to transfection bydigestion with Kpn1 and Pac1, ethanol precipitated and resuspended insterile TE to a concentration of 1.5 mg/mL. Linearized plasmid wasintroduced into exponentially growing Desmond marked cells essentiallyas described, except that 80 μg DNA was used in each electroporation.Forty eight hours postelectroporation, 96 well plates were supplementedwith selection medium—CHO-SSFMII supplemented with 400 μg/mL Geneticin(G418, Gibco/BRL catalog # 10131-019). Plates were maintained inselection medium for up to 30 days, or until cell growth occurred insome of the wells. Such growth was assumed to be the result of clonalexpansion of a single G418 resistant cell. The supernatants from allG418 resistant wells were assayed for C2B8 production by standard ELISAtechniques, and all productive clones were eventually expanded to 120 mLspinner flasks and further analyzed.

Characterization of Antibody Secreting Targeted Cells

A total of 50 electroporations with Molly targeting plasmid were carriedout in this experiment, each of which was plated into separate 96 wellplates. A total of 10 viable, anti-CD20 antibody secreting clones wereobtained and expanded to 120 ml spinner flasks. Genomic DNA was isolatedfrom all clones, and Southern analyses were subsequently performed todetermine whether the clones represented single homologous recombinationevents or whether additional random integrations had occurred in thesame cells. The methods for DNA isolation and Southern hybridizationwere as described in the previous section. Genomic DNA was digested withEcoRI and probed with a PCR generated digoxygenin labelled probe to asegment of the CD20 heavy chain constant region. The results of thisSouthern analysis are presented in FIG. 6. As can be seen in the figure,8 of the 10 clones show a single band hybridizing to the CD20 probe,indicating a single homologous recombination event has occurred in thesecells. Two of the ten, clones 24G2 and 28C9, show the presence ofadditional band(s), indicative of an additional random integrationelsewhere in the genome.

We examined the expression -levels of anti-CD20 antibody in all ten ofthese clones, the data for which is shown in Table 1, below.

TABLE 1 Expression Level of Anti-CD20 Secreting Homologous IntegrantsClone Anti-CD20, pg/c/d 20F4 3.5 25E1 2.4 42F9 1.8 39G11 1.5 21C7 1.350G10 0.9 29F9 0.8 5F9 0.3 28C9* 4.5 24G2* 2.1 *These clones containedadditional randomly integrated copies of anti-CD20. Expression levels ofthese clones therefore reflect a contribution from both the homologousand random sites.Expression levels are reported as picogram per cell per day (pg/c/d)secreted by the individual clones, and represented the mean levelsobtained from three separate ELISAs on samples taken from 120 mL spinnerflasks.

As can be seen from the data, there is a variation in antibody secretionof approximately ten fold between the highest and lowest clones. Thiswas somewhat unexpected as we anticipated similar expression levels fromall clones due to the fact the anti-CD20 genes are all integrated intothe same Desmond marked site. Nevertheless, this observed range inexpression extremely small in comparison to that seen using anytraditional random integration method or with our translationallyimpaired vector system.

Clone 20F4, the highest producing single copy integrant was selected forfurther study. Table 2 (below) presents ELISA and cell culture data fromseven day production runs of this clone.

TABLE 2 7 Day Production Run Data for 20F4 % Viable/ml Day Viable (×10⁵)Tx2 (hr) mg/L pg/c/d 1 96 3.4 31 1.3 4.9 2 94 6 29 2.5 3.4 3 94 9.9 334.7 3.2 4 90 17.4 30 6.8 3 5 73 14 8.3 6 17 3.5 9.5 Clone 20F4 wasseeded at 2 × 10⁵ ml in a 120 ml spinner flask on day 0. On thefollowing six days, cell counts were taken, doubling times calculatedand 1 ml samples of supernatant removed from the flask and analyzed forsecreted anti-CD20 by ELISA.This clone is secreting on average, 3-5 pg antibody/cell/day, based onthis ELISA data. This is the same level as obtained from other highexpressing single copy clones obtained previously in our laboratoryusing the previously developed translationally impaired randomintegration vectors. This result indicates the following:

(1) that the site in the CHO genome marked by the Desmond marking vectoris highly transcriptionally active, and therefore represents anexcellent site from which to express recombinant proteins, and

(2) that targeting by means of homologous recombination can beaccomplished using the subject vectors and occurs at a frequency highenough to make this system a viable and desirable alternative to randomintegration methods.

To further demonstrate the efficacy of this system, we have alsodemonstrated that this site is amplifiable, resulting in even higherlevels of gene expression and protein secretion. Amplification wasachieved by plating serial dilutions of 20F4 cells, starting at adensity of 2.5×10⁴ cells/ml, in 96 well tissue culture dishes, andculturing these cells in media (CHO-SSFMII) supplemented with 5, 10, 15or 20 nM methotrexate. Antibody secreting clones were screened usingstandard ELISA techniques, and the highest producing clones wereexpanded nd further analyzed. A summary of this amplification experimentis presented in Table 3 below.

TABLE 3 Summary of 20F4 Amplification Expression Level # WellsExpression Level # Wells pg/c/d from nM MTX Assayed mg/l 96 wellExpanded spinner 10 56 3-13 4 10-15 15 27 2-14 3 15-18 20 17 4-11 1 NDMethotrexate amplification of 20F4 was set up as described in the text,using the concentrations of methotrexate indicated in the above table.Supernatants from all surviving 96 well colonies were assayed by ELISA,and the range of anti-CD20 expressed by these clones is indicated incolumn 3. Based on these results, the highest producing clones wereexpanded to 120 ml spinners and several ELISAs conducted on the spinnersupernatants to determine the pg/cell/day expression levels, reported incolumn 5.The data here clearly demonstrates that this site can be amplified inthe presence of methotrexate. Clones from the 10 and 15 nMamplifications were found to produce on the order of 15-20 pg/cell/day.

A 15 nM clone, designated 20F4-15A5, was selected as the highestexpressing cell line. This clone originated from a 96 well plate inwhich only 22 wells grew, and was therefore assumed to have arisen froma single cell. A 15 nM clone, designated 20F4-15A5, was selected as thehighest expressing cell line. This clone originated from a 96 well platein which only 22 wells grew, and was therefore assumed to have arisenfrom a single cell. The clone was then subjected to a further round ofmethotrexate amplification. As described above, serial dilutions of theculture were plated into 96 well dishes and cultured in CHO-SS-FMIImedium supplemented with 200, 300 or 400 nM methotrexate. Survivingclones were screened by ELISA, and several high producing clones wereexpanded to spinner cultures and further analyzed. A summary of thissecond amplification experiment is presented in Table 4.

TABLE 4 Summary of 20F4-15A5 Amplification # Wells Expression Level #Wells Expression Level nM MTX Assayed mg/l 96 well Expanded pg/c/d,spinner 200 67 23-70 1 50-60 250 86 21-70 4 55-60 300 81 15-75 3 40-50Methotrexate amplifications of 20F4-15A5 were set up and assayed asdescribed in the text. The highest producing wells, the numbers of whichare indicated in column 4, were expanded to 120 ml spinner flasks. Theexpression levels of the cell lines derived from these wells is recordedas pg/c/d in column 5.The highest producing clone came from the 250 nM methotrexateamplification. The 250 nM clone, 20F4-15A5-250A6 originated from a 96well plate in which only wells grew, and therefore is assumed to havearisen from a single cell. Taken together, the data in Tables 3 and 4strongly indicates that two rounds of methotrexate amplification aresufficient to reach expression levels of 60 pg/cell/day, which isapproaching the maximum secretion capacity of immunoglobulin inmammalian cells (Reff, M. E., Curr. Opin. Biotech., 4:573-576 (1993)).The ability to reach this secretion capacity with just two amplificationsteps further enhances the utility of this homologous recombinationsystem. Typically, random integration methods require more than twoamplification steps to reach this expression level and are generallyless reliable in terms of the ease of amplification. Thus, thehomologous system offers a more efficient and time saving method ofachieving high level gene expression in mammalian cells.

EXAMPLE 5 Expression of Anti-Human CD23 Antibody in Desmond Marked CHOCells

CD23 is low affinity IgE receptor which mediates binding of IgE to B andT lymphocytes (Sutton, B. J., and Gould, H. J., Nature, 366:421-428(1993)). Anti-human CD23 monoclonal antibody 5E8 is a human gamma-1monoclonal antibody recently cloned and expressed in our laboratory.This antibody is disclosed in commonly assigned Ser. No. 08/803,085,filed on Feb. 20, 1997.

The heavy and light chain genes of 5E8 were cloned into the mammalianexpression vector NSKG1, a derivative of the vector NEOSPLA (Barnett etal, in Antibody Expression and Engineering, H. Y Yang and T. Imanaka,eds., pp 27-40 (1995)) and two modifications were then made to thegenes. We have recently observed somewhat higher secretion ofimmunoglobulin light chains compared to heavy chains in other expressionconstructs in the laboratory (Reff et al, 1997, unpublishedobservations). In an attempt to compensate for this deficit, we alteredthe 5E8 heavy chain gene by the addition of a stronger promoter/enhancerelement immediately upstream of the start site. In subsequent steps, a2.9 kb DNA fragment comprising the 5E8 modified light and heavy chaingenes was isolated from the NSKG1 vector and inserted into the targetingvector Mandy. Preparation of 5E8-containing Molly and electroporationinto Desmond 15C9 CHO cells was essentially as described in thepreceding section.

One modification to the previously described protocol was in the type ofculture medium used. Desmond marked CHO cells were cultured inprotein-free CD-CHO medium (Gibco-BRL, catalog # AS21206) supplementedwith 3 mg/L recombinant insulin (3 mg/mL stock, Gibco-BRL, catalog #AS22057) and 8 mM L-glutamine (200 mM stock, Gibco-BRL, catalog #25030-081). Subsequently, transfected cells were selected in the abovemedium supplemented with 400 μg/mL geneticin. In this experiment, 20electroporations were performed and plated into 96 well tissue culturedishes. Cells grew and secreted anti-CD23 in a total of 68 wells, all ofwhich were assumed to be clones originating from a single G418 cell.Twelve of these wells were expanded to 120 ml spinner flasks for furtheranalysis. We believe the increased number of clones isolated in thisexperiment (68 compared with 10 for anti-CD20 as described in Example 4)is due to a higher cloning efficiency and survival rate of cells grownin CD-CHO medium compared with CHO-SS-FMII medium. Expression levels forthose clones analyzed in spinner culture ranged from 0.5-3 pg/c/d, inclose agreement with the levels seen for the anti-CD20 clones. Thehighest producing anti-CD23 clone, designated 4H12, was subjected tomethotrexate amplification in order to increase its expression levels.This amplification was set up in a manner similar to that described forthe anti-CD20 clone in Example 4. Serial dilutions of exponentiallygrowing 4H12 cells were plated into 96 well tissue culture dishes andgrown in CD-CHO medium supplemented with 3 mg/L insulin, 8 mM glutamineand 30, 35 or 40 nM methotrexate. A summary of this amplificationexperiment is presented in Table 5.

TABLE 5 Summary of 2H12 Amplification Expression Level # WellsExpression Level # Wells pg/c/d from nM MTX Assayed mg/l 96 wellExpanded spinner 30 100 6-24 8 10-25 35 64 4-27 2 10-15 40 96 4-20 1 NDThe highest expressing clone obtained was a 30 nM clone, isolated from aplate on which 22 wells had grown. This clone, designated 4H12-30G5, wasreproducibly secreting 18-22 pg antibody per cell per day. This is thesame range of expression seen for the first amplification of the antiCD20 clone 20F4 (clone 20F4-15A5 which produced 15-18 pg/c/d, asdescribed in Example 4). This data serves to further support theobservation that amplification at this marked site in CHO isreproducible and efficient. A second amplification of this 30 nM cellline is currently underway. It is anticipated that saturation levels ofexpression will be achievable for the anti-CD23 antibody in just twoamplification steps, as was the case for anti-CD20.

EXAMPLE 6 Expression of Immunoadhesin in Desmond Marked CHO Cells

CTLA-4, a member of the Ig superfamily, is found on the surface of Tlymphocytes and is thought to play a role in antigen-specific T-cellactivation (Dariavach et al, Eur. J. Immunol., 18:1901-1905 (1988); andLinsley et al, J. Exp. Med., 174:561-569 (1991)). In order to furtherstudy the precise role of the CTLA-4 molecule in the activation pathway,a soluble fusion protein comprising the extracellular domain of CTLA-4linked to a truncated form of the human IgG1 constant region was created(Linsley et al (Id.). We have recently expressed this CTLA-4 Ig fusionprotein in the mammalian expression vector BLECH1, a derivative of theplasmid NEOSPLA (Barnett et al, in Antibody Expression and Engineering,H. Y Yang and T. Imanaka, eds., pp 27-40 (1995)). An 800 bp fragmentencoding the CTLA-4 Ig was isolated from this vector and insertedbetween the SacII and BglII sites in Molly.

Preparation of CTLA-4Ig-Molly and electroporation into Desmond clone15C9 CHO cells was performed as described in the previous examplerelating to anti-CD20. Twenty electroporations were carried out, andplated into 96 well culture dishes as described previously. EighteenCTLA-4 expressing wells were isolated from the 96 well plates andcarried forward to the 120 ml spinner. stage. Southern analyses ongenomic DNA isolated from each of these clones were then carried out todetermine how many of the homologous clones contained additional randomintegrants. Genomic DNA was digested with BglII and probed with a PCRgenerated digoxygenin labelled probe to the human IgC-1 constant region.The results of this analysis indicated that 85% of the CTLA-4 clones arehomologous integrants only; the remaining 15% contained one additionalrandom integrant. This result corroborates the findings from theexpression of anti-CD20 discussed above, where 80% of the clones weresingle homologous integrants. Therefore, we can conclude that thisexpression system reproducibly yields single targeted homologousintegrants in at least 80% of all clones produced.

Expression levels for the homologous CT1A4-Ig clones ranged from 8-12pg/cell/day. This is somewhat higher than the range reported foranti-CD20 antibody and anti-CD23 antibody clones discussed above.However, we have previously observed that expression of this moleculeusing the intronic insertion vector system also resulted insignificantly higher expression levels than are obtained forimmunoglobulins. We are currently unable to provide an explanation forthis observation.

EXAMPLE 7 Targeting Anti-CD20 to an Alternate Desmond Marked CHO CellLine

As we described in a preceding section, we obtained 5 single copyDesmond marked CHO cell lines (see FIGS. 4 and 5.). In order todemonstrate that the success of our targeting strategy is not due tosome unique property of Desmond clone 15C9 and limited only to thisclone, we introduced anti-CD20 Molly into Desmond clone 9B2 (lane 6 inFIG. 4, lane 1 in FIG. 5). Preparation of Molly DNA and electroporationinto Desmond 9B2 was exactly as described in the previous examplepertaining to anti-CD20. We obtained one homologous integrant from thisexperiment. This clone was expanded to a 120 ml spinner flask, where itproduced on average 1.2 pg anti-CD20/cell/day. This is considerablylower expression than we observed with Molly targeted into Desmond 15C9.However, this was the anticipated result, based on our northern analysisof the Desmond clones. As can be seen in FIG. 5, mRNA levels from clone9B2 are considerably lower than those from 15C9, indicating the site inthis clone is not as transcriptionally active as that in 15C9.Therefore, this experiment not only demonstrates the reproducibility ofthe system—presumably any marked Desmond site can be targeted withMolly—it also confirms the northern data that the site in Desmond 15C9is the most transcriptionally active.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without diverting fromthe scope of the invention. Accordingly, the invention is not limited bythe appended claims.

1-41. (canceled)
 42. A method for selecting a mammalian cell that hashigh expression of an immunoglobulin of interest comprising: (i)transfecting mammalian cells with a marking vector, wherein said markingvector comprises (a) a first region of DNA that is heterologous to themammalian cell genome which when integrated in the mammalian cell genomeprovides a unique site for homologous recombination, (b) a first DNAfragment encoding a selectable marker protein that provides forselection of mammalian cells which have been successfully integratedwith the marking vector wherein the selectable marker protein is not aneomycin phosphotransferase, and (c) a second DNA fragment comprising atleast one, but not all, exons of a neomycin phosphotransferase gene;(ii) selecting in vitro for the mammalian cells which contain a singlemarking vector integrated into the genome wherein said cells have highexpression of the selectable marker; (iii) transfecting the mammaliancells that have high expression of the selectable marker with atargeting vector comprising (a) a third DNA fragment comprising theremaining neomycin phosphotransferase exon or exons that are not presentthe marking vector, (b) an immunoglobulin DNA that encodes at least oneimmunoglobulin of interest, and (c) a second region of DNA that isidentical or is sufficiently homologous to the unique site in saidmarking plasmid is integrated into the genome by homologousrecombination at said unique site; and (iv) selecting in vitro for themammalian cells which express said selectable marker, said neomycinphosphotransferase and said immunoglobulin of interest; therebyselecting a mammalian cell that has high expression of saidimmunoglobulin of interest.
 43. The method of claim 42, wherein theselectable marker DNA encodes a protein that is selected from the groupconsisting of histidinol dehydrogenase, dihydrofolate reductase,hygromycin phosphotransferase, herpes simplex virus thymidine kinase,adenosine deaminase, glutamine synthetase, and hypoxanthine guaninephosphoribosyl transferase.
 44. The method of claim 43, wherein theselectable marker DNA encodes a dihydrofolate reductase protein.
 45. Themethod of claim 42, which further comprises determining the RNA levelsof the selectable marker.
 46. The method of claim 42, wherein saidsecond DNA fragment encoding the remaining neomycin phosphotransferaseexons comprise two neomycin phosphotransferase exons which are separatedby the immunoglobulin DNA that encodes at least one immunoglobulin ofinterest.
 47. The method of claim 42, wherein said immunoglobulin DNAencodes a heavy chain and a light chain.
 48. A eukaryotic cell whichcomprises a marking vector integrated into the genome for identifying atranscriptionally active site in the genome of a mammalian cell, whereinsaid marking vector comprises: (i) a first region of DNA that isheterologous to the mammalian cell genome which when integrated in themammalian cell genome provides a unique site for homologousrecombination, (ii) a first selectable marker DNA encoding for a firstselectable marker that provides for selection of mammalian cells whichhave been successfully integrated with the marking vector, and (iii) afirst DNA fragment encoding a portion of a second selectable marker thatis different from the first selectable marker, wherein said secondselectable marker is only expressed in said mammalian cell when atargeting vector comprising (a) a second DNA fragment encoding a secondportion of said second selectable marker DNA and (b) a second region ofDNA that is identical or is sufficiently homologous to the unique sitein said marking plasmid is integrated into the genome by homologousrecombination at said unique site.
 49. The eukaryotic cell of claim 48,wherein said cell is a mammalian cell.
 50. The eukaryotic cell of claim49, wherein said cell is selected from the group consisting of Chinesehamster ovary (CHO) cells, myeloma cells, baby hamster kidney cells, COScells, NSO cells, HeLa cells, and NIH 3T3 cells.
 51. The eukaryotic cellof claim 50, wherein the cell is a CHO cell.
 52. The eukaryotic cell ofclaim 51, wherein the cell is clone 15C9.
 53. A method for marking atranscriptionally active site in the genome of a mammalian cellcomprising: (i) transfecting said mammalian cell with a marking vector,wherein said marking vector comprises (a) a first region of DNA that isheterologous to the mammalian cell genome which when integrated in themammalian cell genome provides a unique site for homologousrecombination, (b) a first selectable marker DNA encoding for a firstselectable marker that provides for selection of mammalian cells whichhave been successfully integrated with the marking vector, and (c) afirst DNA fragment encoding a portion of a second selectable marker thatis different from the first selectable marker, wherein said secondselectable marker is only expressed in said mammalian cell when atargeting vector comprising (a) a second DNA fragment encoding a secondportion of said second selectable marker and (b) a second region of DNAthat is identical or is sufficiently homologous to the unique site insaid marking plasmid is integrated into the genome by homologousrecombination at said unique site; and (ii) selecting in vitro for themammalian cells which contain a single marking vector integrated intothe genome wherein said cells have high expression of said firstselectable marker; thereby marking the transcriptionally active site inthe genome of said mammalian cell.
 54. The method of claim 53, whereinthe in vitro screening comprises determining the protein levels of thefirst selectable marker.
 55. The method of claim 54, wherein the invitro screening further comprises determining the RNA levels of thefirst selectable marker.
 56. The method of claim 53, wherein the firstselectable marker DNA encodes a protein that is selected from the groupconsisting of neomycin phosphotransferase, histidinol dehydrogenase,dihydrofolate reductase, hygromycin phosphotransferase, herpes simplexvirus thymidine kinase, adenosine deaminase, glutamine synthetase, andhypoxanthine guanine phosphoribosyl transferase.
 57. The method of claim56, wherein the first selectable marker DNA encodes a dihydrofolatereductase protein.
 58. The method of claim 53, wherein the secondselectable marker DNA encodes a protein that is selected from the groupconsisting of neomycin phosphotransferase, histidinol dehydrogenase,dihydrofolate reductase, hygromycin phosphotransferase, herpes simplexvirus thymidine kinase, adenosine deaminase, glutamine synthetase, andhypoxanthine guanine phosphoribosyl transferase.
 59. The method of claim58, wherein the second selectable marker DNA encodes a neomycinphosphotransferase protein.
 60. The method of claim 53, wherein the DNAregion that is heterologous to the mammalian cell genome is a bacterialDNA, an insect DNA, a viral DNA or a synthetic DNA.
 61. The method ofclaim 53, wherein the region of DNA that is heterologous to themammalian cell genome does not contain any functional genes.